Aza-Podophyllotoxin analogues as potential anti-cancer agents

ABSTRACT

Novel tetracyclic aza-podophyllotoxin analogue compounds, a method of synthesizing such compounds, a pharmaceutical composition comprising such compounds and a suitable carrier, and a method of using the compounds. The tetracyclic aza-podophyllotoxin analogue compounds are useful as antitumor agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.18/122,047, filed on Mar. 15, 2023, the entire contents of which arehereby incorporated by reference in their entirety.

BACKGROUND 1. Field

The disclosure of the present patent application relates generally tothe clinical application of novel tetracyclic aza-Podophyllotoxinanalogues, for example,benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-ones, as potentialantitumoral/anti-cancer agents.

2. Description of the Related Art

Lignans, a class of natural products found in a variety of plants, areknown to play a crucial role in medicinal chemistry due to theirextensive biological activity and their broad therapeutic applications.The natural aryltetralin lactone podophyllotoxin, a potent inhibitor ofmicrotubule assembly, occupies a unique position among lignans andexhibits both antitumor and antiviral activities. Due to severe toxicityof podophyllotoxin, two synthetic analogues, Etoposide and Tenoposide,are currently in use in cancer chemotherapy. Podophyllotoxins belong toa class of natural products from which the clinically used Etoposide andTenoposide are already known.

Numerous investigations concerning structural modifications ofpodophyllotoxin have been accomplished. In this context a large numberof azapodophyllotoxins derivatives have been synthesized andinvestigated for their antitumour activity (Hua-yang Fan, Zhuo-li Zhu,Hong-chun Xian, Hao-fan Wang, Bing-jun Chen, Ya-Jie Tang, Ya-ling Tang,Xin-hua Liang, Front Cell Dev Biol., 2021; 9: 709075). Tratrat et al.made an important contribution to this field by discovering a greatlysimplified one pot synthesis of azapodophyllotoxine analogues possessingpotent cytotoxicity (Tratrat et al., Org. Lett., 2002, 4, 3187-3189). Inthis context a large number of 4-Aza-2,3-didehydro podophyllotoxinsderivatives have been synthesized and investigated for their antitumouractivity and retain most of the cytotoxicity associated with the parentlignan (M. G. Botes1, S. C. Pelly1, M. A. L. Blackie1, A. Kornienko2,and W. A. L. van Otterlo, Chemistry of Heterocyclic Compounds, 2014, 50(2), 119-138; US 2003/006548515, US 2013/0245048, US 2019/0077808 US2021/0128705). However, none of these analogues have been found to solveall previously known problems with this class of compounds.

It would be desirable to have clinical applications of syntheticanalogues in addition to or instead of the Etoposide and Tenoposide thatare currently in use in cancer chemotherapy, such as derivatives thatdemonstrate good to excellent cytotoxic activity against a large panelof cancer cell lines at a nano-molar concentration range with structuresthat are easily amended to target different drug actions such astopoisomerases, tubulin, and kinases as important molecular targets ofcurrently clinically used anticancer agents.

Thus, new podophyllotoxin compound solving the aforementioned problemsare desired.

SUMMARY

The present subject matter relates to new antitumor agents withpotentially new mechanisms of action alongside those already existingfor a certain class of antitumor agents. Accordingly, the presentsubject matter relates to new aza-podophyllotoxins as potentialanticancer agents.

The present subject matter provides novel tetracyclicaza-podophyllotoxin analogue compounds, for example,benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one compounds of thegeneral formula/structure I, as useful antitumor agents. The presentsubject matter further provides a process for the synthesis ofaza-podophyllotoxin analogue compounds and their use in therapy for thetreatment of cancer as a sole active agent or in combination with otheractive ingredients.

In one embodiment, the present subject matter relates to compounds offormula I:

or a pharmaceutically acceptable salt, ester stereoisomer, or solvatethereof, wherein:

-   -   represents a single or double bond;    -   n is 1 or 2;    -   W represents an oxygen or sulfur;    -   X represents an oxygen, sulfur, CH₂, or —NR₇ wherein R₇        represents a hydrogen atom or a linear or branched (C₁-C₆)alkyl        group, a (C₃-C₉)cycloalkyl group, a (C₁-C₆)alkyl-aryl group, an        aryl group, or a heteroaryl group;    -   R₁ represents:    -   a hydrogen atom,    -   aryl,    -   heteroaryl,    -   (C₃-C₉) cycloalkyl group,    -   linear or branched (C₁-C₆) alkyl group, optionally substituted        by an aryl group, a heteroaryl group, a hydroxy group, a linear        or branched (C₁-C₆) alkoxy group, a carboxylic acid group, a        group of formula —CONR₈R₉    -   or —NR₈R₉, wherein R₈ and R₉, which may be the same or        different, each independently represent a linear or branched        (C₁-C₆)alkyl group optionally substituted by a hydroxy group    -   or an amino group (itself optionally substituted by one or two        linear or branched (C₁-C₆) alkyl groups), or R₈ and R₉, together        with the nitrogen atom to which they are attached, form a        nitrogen-containing heterocycle,    -   an amino group optionally substituted by one or more aryl        groups, heteroaryl groups, or linear or branched (C₁-C₆) alkyl        groups or (C₃-C₆) cycloalkyl groups optionally substituted by a        carboxylic acid group, or by a group of formula —CONR₈R₉ or        —NR₈R₉, or    -   —OR₁₀ wherein R₁₀ represents a hydrogen atom, an aryl group, a        heteroaryl group, or a linear or branched (C₁-C₆) alkyl group or        a (C₃-C₆) cycloalkyl group optionally substituted by a        carboxylic acid group, or by a group of formula —CONR₈R₉ or        —NR₈R₉;    -   R₂, R₃, R₄, and R₅, which may be the same or different, each        represent:        -   a hydrogen atom,        -   a halogen atom,        -   a hydroxy group,        -   a nitro group,        -   a linear or branched (C₁-C₆) polyhaloalkyl group,        -   a linear or branched (C₁-C₆) alkyl group,        -   —OR₁₀,        -   an amino group,            -   a substituted amino group optionally substituted by one                or more aryl groups, heteroaryl groups, or linear or                branched (C₁-C₆) alkyl groups or (C₃-C₆) cycloalkyl                groups optionally substituted by a carboxylic acid                group, by a carbonyl group, or by a group of formula                —CONR₈R₉ or —NR₈R₉,        -   —OPO(OH)₂,        -   an acyl group,        -   a carboxylic acid group,        -   a sulfonyl group,        -   a sulfonamide group, or        -   a cyano group; and    -   R₆ is a hydrogen atom, a linear or branched (C₁-C₆) alkyl group,        a (C₃-C₆) cycloalkyl group, or an aryl or heteroaryl group        optionally substituted in one or more positions with one or more        of R₂, R₃, R₄, and R₅, each of which may be the same or        different.

In a further embodiment, the present subject matter relates to anaza-Podophyllotoxin analogue compound of formula I:

or a pharmaceutically acceptable salt, ester stereoisomer, or solvatethereof, wherein:

-   -   n is 1;    -   W is O;    -   X is O, NH or S;    -   R₁ is H;    -   R₂, R₃, R₄ and R₅ are each H; and        R₆ is phenyl or benzo[d][1,3]dioxolyl, each of which may be        optionally substituted with one or more substituents        independently selected from the group consisting of hydroxy,        methoxy, methyl, N(CH₃)₂, NH₂, —NHCOCH₃, fluorine, bromine, and        chlorine.

In an embodiment, the present subject matter relates to a compoundselected from the group consisting of:

-   11-(4-methoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (1),-   11-(3,4-dimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (2),-   11-(3,4,5-trimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (3),-   11-(benzo[d][1,3]dioxol-5-yl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (4),-   11-(3,4-dihydroxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (5),-   11-(4-hydroxy-3-methoxyphenyl)-4,11-dihydro-1H,3H    benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one (6),-   11-(p-tolyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (7),-   11-(4-(dimethylamino)phenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (8),-   11-(4-aminophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (9),-   N-(4-(1-oxo-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-11-yl)phenyl)acetamide    (10),-   11-(3,4,5-trimethoxyphenyl)-2,3,4,11-tetrahydro-1H-benzo[4,5]imidazo[1,2-a]pyrrolo[3,4-d]pyrimidin-1-one    (11),-   11-(3,4,5-trimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]thieno[3,4-d]pyrimidin-1-one    (12),-   11-(4-fluorophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (13),-   11-(4-chlorophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (14),-   1-(4-bromophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (15),    and a pharmaceutically acceptable salt, ester, stereoisomer, or    solvate thereof.

In an embodiment, the present subject matter relates to a process forthe synthesis of the compounds of formula I, including a number ofspecies or specific structures falling under structural formula I.Further contemplated herein are pharmaceutical compositions containingthese compounds, as well as methods of treating various cancers byadministering the present compounds to a patient in need thereof.

These and other features of the present subject matter will becomereadily apparent upon further review of the following specification.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following definitions are provided for the purpose of understandingthe present subject matter and for construing the appended patentclaims.

Definitions

Throughout the application, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present teachings can alsoconsist essentially of, or consist of, the recited components, and thatthe processes of the present teachings can also consist essentially of,or consist of, the recited process steps.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components. Further, it should be understood that elements and/orfeatures of a composition or a method described herein can be combinedin a variety of ways without departing from the spirit and scope of thepresent teachings, whether explicit or implicit herein.

The use of the terms “include,” “includes”, “including,” “have,” “has,”or “having” should be generally understood as open-ended andnon-limiting unless specifically stated otherwise.

The use of the singular herein includes the plural (and vice versa)unless specifically stated otherwise. In addition, where the use of theterm “about” is before a quantitative value, the present teachings alsoinclude the specific quantitative value itself, unless specificallystated otherwise. As used herein, the term “about” refers to a ±10%variation from the nominal value unless otherwise indicated or inferred.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo, andiodo.

As used herein, “alkyl” refers to a straight-chain or branched saturatedhydrocarbon group. Examples of alkyl groups include methyl (Me), ethyl(Et), propyl (e.g., n-propyl and z′-propyl), butyl (e.g., n-butyl,z′-butyl, sec-butyl, tert-butyl), pentyl groups (e.g., n-pentyl,z′-pentyl, -pentyl), hexyl groups, and the like. In various embodiments,an alkyl group can have 1 to 40 carbon atoms (i.e., C1-C40 alkyl group),for example, 1-30 carbon atoms (i.e., C1-C30 alkyl group). In someembodiments, an alkyl group can have 1 to 6 carbon atoms, and can bereferred to as a “lower alkyl group” or a “C1-C6 alkyl group”. Examplesof lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl andz′-propyl), and butyl groups (e.g., n-butyl, z′-butyl, sec-butyl,tert-butyl). In some embodiments, alkyl groups can be substituted asdescribed herein. An alkyl group is generally not substituted withanother alkyl group, an alkenyl group, or an alkynyl group.

As used herein, “alkenyl” refers to a straight-chain or branched alkylgroup having one or more carbon-carbon double bonds. Examples of alkenylgroups include ethenyl, propenyl, butenyl, pentenyl, hexenyl,butadienyl, pentadienyl, hexadienyl groups, and the like. The one ormore carbon-carbon double bonds can be internal (such as in 2-butene) orterminal (such as in 1-butene). In various embodiments, an alkenyl groupcan have 2 to 40 carbon atoms (i.e., C2-C40 alkenyl group), for example,2 to 20 carbon atoms (i.e., C2-C20 alkenyl group) or 2 to 6 carbon atoms(i.e., C2-C6 alkenyl group). In some embodiments, alkenyl groups can besubstituted as described herein. An alkenyl group is generally notsubstituted with another alkenyl group, an alkyl group, or an alkynylgroup.

The term “substituted alkyl” as used herein refers to an alkyl group inwhich 1 or more (up to about 5, for example about 3) hydrogen atoms isreplaced by a substituent independently selected from the group: —O, —S,acyl, acyloxy, optionally substituted alkoxy, optionally substitutedamino (wherein the amino group may be a cyclic amine), azido, carboxyl,(optionally substituted alkoxy)carbonyl, amido, cyano, optionallysubstituted cycloalkyl, optionally substituted cycloalkenyl, halogen,hydroxyl, nitro, sulfamoyl, sulfanyl, sulfinyl, sulfonyl, and sulfonicacid. Some of the optional substituents for alkyl are hydroxy, halogenexemplified by chloro and bromo, acyl exemplified by methylcarbonyl;alkoxy, and heterocyclyl exemplified by morpholino and piperidino. Otheralkyl substituents as described herein may further be contemplated.

The term “substituted alkenyl” refers to an alkenyl group in which 1 ormore (up to about 5, for example about 3) hydrogen atoms is replaced bya substituent independently selected from those listed above withrespect to a substituted alkyl. Other alkenyl substituents as describedherein may further be contemplated.

As used herein, “heteroatom” refers to an atom of any element other thancarbon or hydrogen and includes, for example, nitrogen, oxygen, silicon,sulfur, phosphorus, and selenium.

As used herein, “aryl” refers to an aromatic monocyclic hydrocarbon ringsystem or a polycyclic ring system in which two or more aromatichydrocarbon rings are fused (i.e., having a bond in common with)together or at least one aromatic monocyclic hydrocarbon ring is fusedto one or more cycloalkyl and/or cycloheteroalkyl rings. An aryl groupcan have 6 to 24 carbon atoms in its ring system (e.g., C6-C24 arylgroup), which can include multiple fused rings. In some embodiments, apolycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ringposition of the aryl group can be covalently linked to the definedchemical structure. Examples of aryl groups having only aromaticcarbocyclic ring(s) include phenyl, 1-naphthyl (bicyclic), 2-naphthyl(bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic),pentacenyl (pentacyclic), and like groups. Examples of polycyclic ringsystems in which at least one aromatic carbocyclic ring is fused to oneor more cycloalkyl and/or cycloheteroalkyl rings include, among others,benzo derivatives of cyclopentane (i.e., an indanyl group, which is a5,6-bicyclic cycloalkyl/aromatic ring system), cyclohexane (i.e., atetrahydronaphthyl group, which is a 6,6-bicyclic cycloalkyl/aromaticring system), imidazoline (i.e., a benzimidazolinyl group, which is a5,6-bicyclic cycloheteroalkyl/aromatic ring system), and pyran (i.e., achromenyl group, which is a 6,6-bicyclic cycloheteroalkyl/aromatic ringsystem). Other examples of aryl groups include benzodioxanyl,benzodioxolyl, chromanyl, indolinyl groups, and the like. In someembodiments, aryl groups can be substituted as described herein. In someembodiments, an aryl group can have one or more halogen substituents,and can be referred to as a “haloaryl” group. Perhaloaryl groups, i.e.,aryl groups where all of the hydrogen atoms are replaced with halogenatoms (e.g., —C6F5), are included within the definition of “haloaryl”.In certain embodiments, an aryl group is substituted with another arylgroup and can be referred to as a biaryl group. Each of the aryl groupsin the biaryl group can be substituted as disclosed herein.

As used herein, “heteroaryl” refers to an aromatic monocyclic ringsystem containing at least one ring heteroatom selected from oxygen (O),nitrogen (N), sulfur (S), silicon (Si), and selenium (Se) or apolycyclic ring system where at least one of the rings present in thering system is aromatic and contains at least one ring heteroatom.Polycyclic heteroaryl groups include those having two or more heteroarylrings fused together, as well as those having at least one monocyclicheteroaryl ring fused to one or more aromatic carbocyclic rings,non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkylrings. A heteroaryl group, as a whole, can have, for example, 5 to 24ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 memberedheteroaryl group). The heteroaryl group can be attached to the definedchemical structure at any heteroatom or carbon atom that results in astable structure. Generally, heteroaryl rings do not contain O—O, S—S,or S—O bonds. However, one or more N or S atoms in a heteroaryl groupcan be oxidized (e.g., pyridine N-oxide thiophene S-oxide, thiopheneS,S-dioxide). Examples of heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below:where T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g., N-benzyl),SiH2, SiH(alkyl), Si(alkyl)2, SiH(arylalkyl), Si(arylalkyl)2, orSi(alkyl)(arylalkyl). Examples of such heteroaryl rings includepyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl,thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl,benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl,quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl,benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl,cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuryl,naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl,thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl,pyridopyrimidinyl, pyridopyrazinyl, pyridopyrdazinyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl groups, and the like. Further examplesof heteroaryl groups include 4,5,6,7-tetrahydroindolyl,tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups,and the like. In some embodiments, heteroaryl groups can be substitutedas described herein.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined herein.

It will be understood by those skilled in the art with respect to anychemical group containing one or more substituents that such groups arenot intended to introduce any substitution or substitution patterns thatare sterically impractical and/or physically non-feasible.

The term “isomers” or “stereoisomers” as used herein relates tocompounds that have identical molecular formulae but that differ in thearrangement of their atoms in space. Stereoisomers that are not mirrorimages of one another are termed “diastereoisomers” and stereoisomersthat are non-superimposable mirror images are termed “enantiomers,” orsometimes optical isomers. A carbon atom bonded to four non-identicalsubstituents is termed a “chiral center.” Certain compounds herein haveone or more chiral centers and therefore may exist as either individualstereoisomers or as a mixture of stereoisomers. Configurations ofstereoisomers that owe their existence to hindered rotation about doublebonds are differentiated by their prefixes cis and trans (or Z and E),which indicate that the groups are on the same side (cis or Z) or onopposite sides (trans or E) of the double bond in the molecule accordingto the Cahn-Ingold-Prelog rules. All possible stereoisomers arecontemplated herein as individual stereoisomers or as a mixture ofstereoisomers.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges,percentage ranges, or ratio ranges, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the described subject matter. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and such embodiments are alsoencompassed within the described subject matter, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use“comprising” language. However, it will be understood by one of skill inthe art, that in some specific instances, an embodiment canalternatively be described using the language “consisting essentiallyof” or “consisting of”.

“Subject” as used herein refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, and petcompanion animals such as household pets and other domesticated animalssuch as, but not limited to, cattle, sheep, ferrets, swine, horses,poultry, rabbits, goats, dogs, cats and the like.

“Patient” as used herein refers to a subject in need of treatment of acondition, disorder, or disease, such as an acute or chronic airwaydisorder or disease.

For purposes of better understanding the present teachings and in no waylimiting the scope of the teachings, unless otherwise indicated, allnumbers expressing quantities, percentages or proportions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, each numerical parametershould at least be construed in light of the number of reportedsignificant digits and by applying ordinary rounding techniques.

In the instant application the terms“benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one derivatives”,“tetracyclic aza-Podophyllotoxin analogues ofbenzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one”,“aza-Podophyllotoxin analogue compounds”,“benzimidazofuropyrimidin-1-one derivatives”, and the like may be usedinterchangeably.

Aza-Podophyllotoxin Analogue Compounds

In one embodiment, the present subject matter may includeaza-Podophyllotoxin analogue compounds having the Structure I:

or a pharmaceutically acceptable salt, ester stereoisomer, or solvatethereof, wherein:

-   -   represents a single or double bond;    -   n is 1 or 2;    -   W represents an oxygen or sulfur;    -   X represents an oxygen, sulfur, CH₂, or —NR₇ wherein R₇        represents a hydrogen atom or a linear or branched (C₁-C₆)alkyl        group, a (C₃-C₉)cycloalkyl group, a (C₁-C₆)alkyl-aryl group, an        aryl group, or a heteroaryl group;    -   R₁ represents:        -   a hydrogen atom,        -   aryl,        -   heteroaryl,        -   (C₃-C₉) cycloalkyl group,        -   linear or branched (C₁-C₆) alkyl group, optionally            substituted by an aryl group, a heteroaryl group, a hydroxy            group, a linear or branched (C₁-C₆) alkoxy group, a            carboxylic acid group, a group of formula —CONR₈R₉ or            —NR₈R₉, wherein R₈ and R₉, which may be the same or            different, each independently represent a linear or branched            (C₁-C₆)alkyl group optionally substituted by a hydroxy group    -   or an amino group (itself optionally substituted by one or two        linear or branched (C₁-C₆) alkyl groups), or R₈ and R₉, together        with the nitrogen atom to which they are attached, form a        nitrogen-containing heterocycle,        -   an amino group optionally substituted by one or more aryl            groups, heteroaryl groups, or linear or branched (C₁-C₆)            alkyl groups or (C₃-C₆) cycloalkyl groups optionally            substituted by a carboxylic acid group, or by a group of            formula —CONR₈R₉ or —NR₈R₉, or        -   —OR₁₀ wherein R₁₀ represents a hydrogen atom, an aryl group,            a heteroaryl group, or a linear or branched (C₁-C₆) alkyl            group or a (C₃-C₆) cycloalkyl group optionally substituted            by a carboxylic acid group, or by a group of formula            —CONR₈R₉ or —NR₈R₉;    -   R₂, R₃, R₄, and R₅, which may be the same or different, each        represent:        -   a hydrogen atom,        -   a halogen atom,        -   a hydroxy group,        -   a nitro group,        -   a linear or branched (C₁-C₆) polyhaloalkyl group,        -   a linear or branched (C₁-C₆) alkyl group,        -   —OR₁₀,        -   an amino group,        -   a substituted amino group optionally substituted by one or            more aryl groups, heteroaryl groups, or linear or branched            (C₁-C₆) alkyl groups or (C₃-C₆) cycloalkyl groups optionally            substituted by a carboxylic acid group, by a carbonyl group,            or by a group of formula —CONR₈R₉ or —NR₈R₉,        -   —OPO(OH)₂,        -   an acyl group,        -   a carboxylic acid group,        -   a sulfonyl group,        -   a sulfonamide group, or        -   a cyano group; and    -   R₆ is a hydrogen atom, a linear or branched (C₁-C₆) alkyl group,        a (C₃-C₆) cycloalkyl group, or an aryl or heteroaryl group        optionally substituted in one or more positions with one or more        of R₂, R₃, R₄, and R₅, each of which may be the same or        different.

In a further embodiment, the present subject matter relates to anaza-Podophyllotoxin analogue compound of formula I, wherein n is 1.

In another embodiment, the present subject matter relates to compoundsof formula I, wherein W is O.

In yet another embodiment, the present subject matter relates tocompounds of formula I, wherein X is S, NH, or O.

In still yet another embodiment, the present subject matter relates tocompounds of formula I, wherein R₁ is H.

In a further embodiment, the present subject matter relates to compoundsof formula I, wherein R₂, R₃, R₄, and R₅ are each H.

In another embodiment, the present subject matter relates to a compoundof formula I, wherein R₆ is phenyl or benzo[d][1,3]dioxolyl, each ofwhich may be optionally substituted with one or more substituentsindependently selected from the group consisting of hydroxy, methoxy,methyl, N(CH₃)₂, NH₂, —NHCOCH₃, fluorine, bromine, and chlorine. In oneembodiment in this regard, R₆ is phenyl substituted by 1, 2, or 3methoxy groups. In another embodiment in this regard, R₆ is phenylsubstituted by 1 or 2 hydroxy groups, or by one hydroxy group and onemethyl group.

In an embodiment, the present subject matter relates to a compoundselected from the group consisting of:

-   11-(4-methoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (1),-   11-(3,4-dimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (2),-   11-(3,4,5-trimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (3),-   11-(benzo[d][1,3]dioxol-5-yl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (4),-   11-(3,4-dihydroxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (5),-   11-(4-hydroxy-3-methoxyphenyl)-4,11-dihydro-1H,3H    benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one (6),-   11-(p-tolyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (7),-   11-(4-(dimethylamino)phenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (8),-   11-(4-aminophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (9),-   N-(4-(1-oxo-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-11-yl)phenyl)acetamide    (10),-   11-(3,4,5-trimethoxyphenyl)-2,3,4,11-tetrahydro-1H-benzo[4,5]imidazo[1,2-a]pyrrolo[3,4-d]pyrimidin-1-one    (11),-   11-(3,4,5-trimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]thieno[3,4-d]pyrimidin-1-one    (12),-   11-(4-fluorophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (13),-   11-(4-chlorophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (14),-   11-(4-bromophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one    (15),    and a pharmaceutically acceptable salt, ester stereoisomer, or    solvate thereof.

In the present subject matter, the structures of the above compounds areillustrated as follows:

and a pharmaceutically acceptable salt, ester, stereoisomer, or solvatethereof.

It is to be understood that the present subject matter covers allcombinations of substituent groups referred to herein.

The present compounds may contain, e.g., when isolated in crystallineform, varying amounts of solvents. Accordingly, the present subjectmatter includes all solvates of the present compounds of formula I andpharmaceutically acceptable stereoisomers, esters, and/or salts thereof.Hydrates are one example of such solvates.

Further, the present subject matter includes all mixtures of possiblestereoisomers of the embodied compounds, independent of the ratio,including the racemates.

Salts of the present compounds, or the salts of the stereoisomersthereof, include all inorganic and organic acid addition salts and saltswith bases, especially all pharmaceutically acceptable inorganic andorganic acid addition salts and salts with bases, particularly allpharmaceutically acceptable inorganic and organic acid addition saltsand salts with bases customarily used in pharmacy.

Examples of acid addition salts include, but are not limited to,hydrochlorides, hydrobromides, phosphates, nitrates, sulfates, acetates,trifluoroacetates, citrates, D-gluconates, benzoates,2-(4-hydroxy-benzoyl)benzoates, butyrates, sub salicylates, maleates,laurates, malates, lactates, fumarates, succinates, oxalates, tartrates,stearates, benzenesulfonates (besilates), toluenesulfonates (tosilates),methanesulfonates (mesilates) and 3-hydroxy-2-naphthoates.

Examples of salts with bases include, but are not limited to, lithium,sodium, potassium, calcium, aluminum, magnesium, titanium, ammonium,meglumine and guanidinium salts. The salts include water-insoluble and,particularly, water-soluble salts.

The present compounds, the salts, the stereoisomers and the salts of thestereoisomers thereof may contain, e.g., when isolated in crystallineform, varying amounts of solvents. Included within the present scopeare, therefore, all solvates of the compounds of formula I, as well asthe solvates of the salts, the stereoisomers and the salts of thestereoisomers of the compounds of formula I.

The present compounds may be isolated and purified in a manner known perse, e.g., by distilling off the solvent in vacuo and recrystallizing theresidue obtained from a suitable solvent or subjecting it to one of thecustomary purification methods, such as column chromatography on asuitable support material.

Salts of the compounds of formula I and the stereoisomers thereof can beobtained by dissolving the free compound in a suitable solvent (by wayof non-limiting example, a ketone such as acetone, methylethylketone ormethylisobutylketone; an ether such as diethyl ether, tetrahydrofuraneor dioxane; a chlorinated hydrocarbon such as methylene chloride orchloroform; a low molecular weight aliphatic alcohol such as methanol,ethanol or isopropanol; a low molecular weight aliphatic ester such asethyl acetate or isopropyl acetate; or water) which contains the desiredacid or base, or to which the desired acid or base is then added. Theacid or base can be employed in salt preparation, depending on whether amono- or polybasic acid or base is concerned and depending on which saltis desired, in an equimolar quantitative ratio or one differingtherefrom. The salts are obtained by filtering, reprecipitating,precipitating with a non-solvent for the salt or by evaporating thesolvent. Salts obtained can be converted into the free compounds which,in turn, can be converted into salts. In this manner, pharmaceuticallyunacceptable salts, which can be obtained, for example, as processproducts in the manufacturing on an industrial scale, can be convertedinto pharmaceutically acceptable salts by processes known to the personskilled in the art.

Pure diastereomers and pure enantiomers of the present compounds can beobtained, e.g., by asymmetric synthesis, by using chiral startingcompounds in synthesis and by splitting up enantiomeric anddiastereomeric mixtures obtained in synthesis. Preferably, the purediastereomeric and pure enantiomeric compounds are obtained by usingchiral starting compounds in synthesis.

Enantiomeric and diastereomeric mixtures can be split up into the pureenantiomers and pure diastereomers by methods known to a person skilledin the art. Preferably, diastereomeric mixtures are separated bycrystallization, in particular fractional crystallization, orchromatography. Enantiomeric mixtures can be separated, e.g., by formingdiastereomers with a chiral auxiliary agent, resolving the diastereomersobtained and removing the chiral auxiliary agent. As chiral auxiliaryagents, for example, chiral acids can be used to separate enantiomericbases and chiral bases can be used to separate enantiomeric acids viaformation of diastereomeric salts. Furthermore, diastereomericderivatives such as diastereomeric esters can be formed fromenantiomeric mixtures of alcohols or enantiomeric mixtures of acids,respectively, using chiral acids or chiral alcohols, respectively, aschiral auxiliary agents. Additionally, diastereomeric complexes ordiastereomeric clathrates may be used for separating enantiomericmixtures. Alternatively, enantiomeric mixtures can be split up usingchiral separating columns in chromatography. Another suitable method forthe isolation of enantiomers is enzymatic separation.

Synthesis:

In one embodiment, the present subject matter further relates to aprocess for synthesizing the aza-podophyllotoxin analogue compounds offormula/structure I, the process comprising the following generalreaction scheme:

The synthesis of the derivatives of the current subject matter ofstructure I relies on a one pot multiple component reaction by usingdiverse aldehydes, 2-aminobenzimidazole and activated methylenederivative such as tetronic acid.

Pharmaceutical Compositions:

In another embodiment, the present subject matter is directed topharmaceutical compositions comprising a therapeutically effectiveamount of the compounds as described herein together with one or morepharmaceutically acceptable carriers, excipients, or vehicles. In someembodiments, the present compositions can be used for combinationtherapy, where other therapeutic and/or prophylactic ingredients can beincluded therein.

The present subject matter further relates to a pharmaceuticalcomposition, which comprises at least one of the present compoundstogether with at least one pharmaceutically acceptable auxiliary.

In an embodiment, the pharmaceutical composition comprises one or two ofthe present compounds, or one of the present compounds.

Non-limiting examples of suitable excipients, carriers, or vehiclesuseful herein include liquids such as water, saline, glycerol,polyethylene glycol, hyaluronic acid, ethanol, and the like. Suitableexcipients for nonliquid formulations are also known to those of skillin the art. A thorough discussion of pharmaceutically acceptableexcipients and salts useful herein is available in Remington'sPharmaceutical Sciences, 18th Edition. Easton, Pa., Mack PublishingCompany, 1990, the entire contents of which are incorporated byreference herein.

The present compounds are typically administered at a therapeutically orpharmaceutically effective dosage, e.g., a dosage sufficient to providetreatment for an acute or chronic airway disease or disorder.Administration of the compounds or pharmaceutical compositions thereofcan be by any method that delivers the compounds systemically and/orlocally. These methods include oral routes, parenteral routes,intraduodenal routes, and the like.

While human dosage levels have yet to be optimized for the presentcompounds, generally, a daily dose is from about 0.01 to 10.0 mg/kg ofbody weight, for example about 0.1 to 5.0 mg/kg of body weight. Theprecise effective amount will vary from subject to subject and willdepend upon the species, age, the subject's size and health, the natureand extent of the condition being treated, recommendations of thetreating physician, and the therapeutics or combination of therapeuticsselected for administration. The subject may be administered as manydoses as is required to reduce and/or alleviate the signs, symptoms, orcauses of the disease or disorder in question, or bring about any otherdesired alteration of a biological system.

In employing the present compounds for treatment of a bacterialinfection, any pharmaceutically acceptable mode of administration can beused with other pharmaceutically acceptable excipients, including solid,semi-solid, liquid or aerosol dosage forms, such as, for example,tablets, capsules, powders, liquids, suspensions, suppositories,aerosols or the like. The present compounds can also be administered insustained or controlled release dosage forms, including depotinjections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for the prolongedadministration of the compound at a predetermined rate, preferably inunit dosage forms suitable for single administration of precise dosages.

The present compounds may also be administered as compositions preparedas foods for foods or animals, including medical foods, functional food,special nutrition foods and dietary supplements. A “medical food” is aproduct prescribed by a physician that is intended for the specificdietary management of a disorder or health condition for whichdistinctive nutritional requirements exist and may include formulationsfed through a feeding tube (referred to as enteral administration orgavage administration).

A “dietary supplement” shall mean a product that is intended tosupplement the human diet and may be provided in the form of a pill,capsule, tablet, or like formulation. By way of non-limiting example, adietary supplement may include one or more of the following dietaryingredients: vitamins, minerals, herbs, botanicals, amino acids, anddietary substances intended to supplement the diet by increasing totaldietary intake, or a concentrate, metabolite, constituent, extract, orcombinations of these ingredients, not intended as a conventional foodor as the sole item of a meal or diet. Dietary supplements may also beincorporated into foodstuffs, such as functional foods designed topromote control of glucose levels. A “functional food” is an ordinaryfood that has one or more components or ingredients incorporated into itto give a specific medical or physiological benefit, other than a purelynutritional effect. “Special nutrition food” means ingredients designedfor a particular diet related to conditions or to support treatment ofnutritional deficiencies.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,for example about 0.5% to 50%, by weight of a compound or salt of thepresent compounds, the remainder being suitable pharmaceuticalexcipients, carriers, etc.

One manner of administration for the conditions detailed above is oral,using a convenient daily dosage regimen which can be adjusted accordingto the degree of affliction. For such oral administration, apharmaceutically acceptable, non-toxic composition is formed by theincorporation of any of the normally employed excipients, such as, forexample, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and the like. Such compositions take theform of solutions, suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations and the like.

The present compositions may take the form of a pill or tablet and thusthe composition may contain, along with the active ingredient, a diluentsuch as lactose, sucrose, dicalcium phosphate, or the like; a lubricantsuch as magnesium stearate or the like; and a binder such as starch, gumacacia, polyvinylpyrrolidine, gelatin, cellulose and derivativesthereof, and the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier, such as, forexample, water, saline, aqueous dextrose, glycerol, glycols, ethanol,and the like, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, or solubilizing agents, pH buffering agents and thelike, for example, sodium acetate, sodium citrate, cyclodextrinederivatives, sorbitan monolaurate, triethanolamine acetate,triethanolamine oleate, etc.

For oral administration, a pharmaceutically acceptable non-toxiccomposition may be formed by the incorporation of any normally employedexcipients, such as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, talcum, cellulose derivatives,sodium crosscarmellose, glucose, sucrose, magnesium carbonate, sodiumsaccharin, talcum and the like. Such compositions take the form ofsolutions, suspensions, tablets, capsules, powders, sustained releaseformulations and the like.

For a solid dosage form, a solution or suspension in, for example,propylene carbonate, vegetable oils or triglycerides, may beencapsulated in a gelatin capsule. Such diester solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545, the contents of each of which areincorporated herein by reference. For a liquid dosage form, thesolution, e.g., in a polyethylene glycol, may be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and the like, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

Other useful formulations include those set forth in U.S. Pat. Nos. Re.28,819 and 4,358,603, the contents of each of which are herebyincorporated by reference.

Another manner of administration is parenteral administration, generallycharacterized by injection, either subcutaneously, intramuscularly orintravenously. Injectables can be prepared in conventional forms, eitheras liquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,solubility enhancers, and the like, such as for example, sodium acetate,sorbitan monolaurate, triethanolamine oleate, cyclodextrins, etc.

Another approach for parenteral administration employs the implantationof a slow-release or sustained-release system, such that a constantlevel of dosage is maintained. The percentage of active compoundcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the compound and theneeds of the subject. However, percentages of active ingredient of 0.01%to 10% in solution are employable and will be higher if the compositionis a solid which will be subsequently diluted to the above percentages.The composition may comprise 0.2% to 2% of the active agent in solution.

Nasal solutions of the active compound alone or in combination withother pharmaceutically acceptable excipients can also be administered.

Formulations of the active compound or a salt may also be administeredto the respiratory tract as an aerosol or solution for a nebulizer, oras a microtine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation have diameters of less than 50 microns, for example lessthan 10 microns.

Methods of Use:

The present compounds have valuable pharmaceutical properties, whichmake them commercially utilizable. Accordingly, the present subjectmatter further relates to use of the present compounds for the treatmentof diseases such as cancers.

In another embodiment of the present subject matter, the aforementionedcompound derivatives demonstrated in vitro anticancer action againsthuman cancer cell lines comprising MCF-7 (breast cancer), A-549 (lungcancer), and HL-60 (leukemia). Accordingly, the present subject matterrelates to methods of treating a cancer in a patient by administeringone or more of the compounds presented herein to a patient in needthereof. In certain embodiments, the cancer is breast cancer, lungcancer, or leukemia.

In another embodiment of the present subject matter, the concentrationof the present compounds engaged for in vitro study against lung cancercell lines for IC50 was in the range of about 0.058 to 0.211 μM at anexposure period of at least 48 hrs.

In another embodiment of the present subject matter, the concentrationof the present compounds engaged for in vitro study against breastcancer cell lines for IC50 was in the range of about 0.045 to 0.374 μMat an exposure period of at least 48 hrs.

In another embodiment of the present subject matter, the concentrationof the present compounds engaged for in vitro study against leukemiacell lines for IC50 was in the range of 0.061 to 0.281 μM at an exposureperiod of at least 48 hrs.

Pharmacological Activity

The cytotoxicity activity of the compounds of the current subject matterwas assessed against three cancer cell lines such as A-549 (lungcancer), MCF-7 (breast cancer) and HL-60 (leukemia) cancer cells. Thebiological results, reported in Table 1, demonstrated that all thederivatives of Formula I displayed potent in vitro anti-proliferativeactivity with nanomolar concentration on all cancer cell lines rangingfrom 45 nM to 374 nM. By way of example, the compound of Example 13showed one of the most potent cytotoxic derivatives in the series withIC₅₀ (concentration of cytotoxic agent which inhibits proliferation ofthe treated cells by 50%) of 71 nM, 45 nM and 61 nM against A-549, MCF-7and HL-60 cancer cells respectively. In conclusion, thebenzimidazofuropyrimidin-1-one derivatives of the current subject mattershowing highly potent cytotoxic agents may find valuable application intreating a variety of cancers either as a sole active agent or incombination with other active ingredients.

In Vitro Cytotoxic Activity Assay

Compounds 1-15 were screened for their in vitro cytotoxic behaviorutilizing a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay against selected cancer human cell lines consistingof MCF-7 (human breast cancer), A-549 (lung cancer) and HL-60 (leukemia)(T. Mosmann, J. Immunol. Meth., 1983, 65, 55-63). The cells werecultured at 37° C. in RMPI1640 medium supplemented with 10% fetal bovineserum, 50 IU/mL penicillin, and 50 μg/mL streptomycin in a 5% CO2incubator. All cells were sub-cultured 3 times/week by trypsinization.Viable cells were seeded and allowed to adhere for 12 h before a testdrug was added in 96-well plates at an initial density of 1.0×105cells/mL. Tumour cell lines were separately exposed to variousconcentrations of the tested compounds followed by incubation at atemperature of 37° C. during 24 h inside a medium of fresh RMPI 1640.Cells were subsequently incubated at 37° C. using MTT at 0.5 mg/mLduring 4 h. After removal of supernatant, formazan crystals weredissolved in isopropanol and the optical density was measured at 570 nm.Etopside and Cisplatin were used as a positive control. The results aresummarized with Etopside and Cisplatin as standard drugs in Table 1.

TABLE 1 In vitro cytotoxic activity (IC₅₀, μM) ofbenzimidazofuropyrimidinones 1-15 Anti-proliferative activity: IC₅₀ (μM)Entry A-549 MCF-7 HL-60 1 0.092 0.126 0.082 2 0.120 0.104 0.096 3 0.0810.115 0.128 4 0.104 0.097 0.084 5 0.151 0.154 0.178 6 0.095 0.105 0.1337 0.067 0.069 0.072 8 0.092 0.087 0.129 9 0.123 0.162 0.143 10 0.2110.374 0.281 11 0.086 0.125 0.104 12 0.075 0.111 0.085 13 0.071 0.0450.061 14 0.058 0.074 0.069 15 0.105 0.081 0.092 Etoposide 11.92 32.810.31 Cisplatin 9.25 15.86 1.16 ^(a)Cancer cell lines: A-549 (lungcancer), MCF-7 (breast cancer) and HL-60 (leukemia).

The biological results demonstrated that the compounds of Formula Idisplayed potent in vitro anti-proliferative activity against cancercell lines as compared to the control drugs.

The following examples relate to various methods of manufacturingcertain specific compounds as described herein.

EXAMPLES Example 111-(4-methoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(1)

To a mixture of 2-aminobenzimidazole (1 mmol), 4-methoxybenzaldehyde(1.2 mmol), tetronic acid (1.2 mmol) and 10% L-proline (10 mg) in 5 mLethanol were heated at reflux for 1 hour. After cooling at roomtemperature, the precipitate obtained was filtered off, washed withethanol and then recrystallized to yield the expected product.

Elemental Analysis: Calculated: C, 68.46; H, 4.54; N, 12.61. Found: C,68.35; H, 4.66; N, 12.72.

Example 211-(3,4-dimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(2)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,3,4-dimethoxybenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 66.11; H, 4.72; N, 11.56. Found C,66.04; H, 4.77; N, 11.48.

Example 311-(3,4,5-trimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(3)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,3,4,5-trimethoxybenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 64.12; H, 4.87; N, 10.68. Found C,64.31; H, 4.78; N, 10.55.

Example 411-(benzo[d][1,3]dioxol-5-yl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(4)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole, piperonaland tetronic acid.

Elemental Analysis: Calculated C, 65.70; H, 3.77; N, 12.10. Found C,65.89; H, 3.75; N, 12.02.

Example 511-(3,4-dihydroxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(5)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,3,4-dihydroxybenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 64.48; H, 3.91; N, 12.53. Found C,64.29; H, 3.82; N, 12.61.

Example 611-(4-hydroxy-3-methoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(6)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,4-hydroxy-3-methoxybenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 65.32; H, 4.33; N, 12.03. Found C,65.57; H, 4.29; N, 11.88.

Example 711-(p-tolyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(7)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,p-tolualdehyde and tetronic acid. Elemental Analysis:

Calculated C, 71.91; H, 4.76; N, 13.24. Found C, 70.03; H, 4.61; N,13.35.

Example 811-(4-(dimethylamino)phenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(8)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,4-dimethylaminobenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 69.35; H, 5.24; N, 16.17. Found C,69.06; H, 5.18; N, 16.09.

Example 911-(4-aminophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(9)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,4-aminobenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 67.92; H, 4.43; N, 17.60. Found C,68.15; H, 4.37; N, 17.66.

Example 10N-(4-(1-oxo-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-11-yl)phenyl)acetamide(10)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,N-(4-formylphenyl)acetamide and tetronic acid.

Elemental Analysis: Calculated C, 66.66; H, 4.48; N, 15.55. Found C,66.48; H, 4.39; N, 15.61.

Example 1111-(3,4,5-trimethoxyphenyl)-2,3,4,11-tetrahydro-1H-benzo[4,5]imidazo[1,2-a]pyrrolo[3,4-d]pyrimidin-1-one(11)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,3,4,5-trimethoxybenzaldehyde and pyrrolidine-2,4-dione.

Elemental Analysis: Calculated C, 64.28; H, 5.14; N, 14.28. Found C,64.19; H, 5.06; N, 14.32.

Example 1211-(3,4,5-trimethoxyphenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]thieno[3,4-d]pyrimidin-1-one(12)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,3,4,5-trimethoxybenzaldehyde and thiotetronic acid.

Elemental Analysis: Calculated C, 61.60; H, 4.68; N, 10.26. Found C,61.48; H, 4.73; N, 10.31.

Example 1311-(4-fluorophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(13)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,4-fluorobenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 67.29; H, 3.76; N, 13.08. Found C,67.44; H, 3.64; N, 13.12.

Example 1411-(4-chlorophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(14)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,4-chlorobenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 64.01; H, 3.58; N, 12.44. Found C,63.87; H, 3.52; N, 12.53.

Example 1511-(4-bromophenyl)-4,11-dihydro-1H,3H-benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one(15)

The expected product is obtained in accordance with the compounddescribed in Example 1, starting from 2-aminobenzimidazole,4-bromobenzaldehyde and tetronic acid.

Elemental Analysis: Calculated C, 56.56; H, 3.16; N, 10.99. Found C,56.39; H, 3.28; N, 11.14.

It is to be understood that the tetracyclic aza-Podophyllotoxinanalogues, for example,benzo[4,5]imidazo[1,2-a]furo[3,4-d]pyrimidin-1-one compounds,use/application, method of making, and properties of said compounds arenot limited to the specific embodiments or examples described above, butencompasses any and all embodiments within the scope of the genericlanguage of the following claims enabled by the embodiments describedherein, or otherwise shown in the drawings or described above in termssufficient to enable one of ordinary skill in the art to make and usethe instant subject matter compounds.

We claim:
 1. A method of treating a cancer in a patient, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound having the formula I:

or a pharmaceutically acceptable salt, ester, stereoisomer, or solvatethereof, wherein

represents a single or double bond, n is 1; W represents oxygen (O) orsulfur (S); X represents an oxygen, sulfur, CH₂, or —NR₇ wherein R₇represents a hydrogen atom or a linear or branched (C₁-C₆)alkyl group, a(C₃-C₉)cycloalkyl group, a (C₁-C₆)alkyl-aryl group, an aryl group, or aheteroaryl group; R₁ represents: a hydrogen atom, aryl, heteroaryl,(C₃-C₉) cycloalkyl group, linear or branched (C₁-C₆) alkyl group,optionally substituted by an aryl group, a heteroaryl group, a hydroxygroup, a linear or branched (C₁-C₆) alkoxy group, a carboxylic acidgroup, a group of formula —CONR₈R₉ or —NR₈R₉, wherein R₈ and R₉, whichmay be the same or different, each independently represent a linear orbranched (C₁-C₆)alkyl group optionally substituted by a hydroxy group oran amino group (itself optionally substituted by one or two linear orbranched (C₁-C₆) alkyl groups), or R₈ and R₉, together with the nitrogenatom to which they are attached, form a nitrogen-containing heterocycle,an amino group optionally substituted by one or more aryl groups,heteroaryl groups, or linear or branched (C₁-C₆) alkyl groups or (C₃-C₆)cycloalkyl groups optionally substituted by a carboxylic acid group, orby a group of formula —CONR₈R₉ or —NR₈R₉, or —OR₁₀ wherein R₁₀represents a hydrogen atom, an aryl group, a heteroaryl group, or alinear or branched (C₁-C₆) alkyl group or a (C₃-C₆) cycloalkyl groupoptionally substituted by a carboxylic acid group, or by a group offormula —CONR₈R₉ or —NR₈R₉; R₂, R₃, R₄, and R₅, which may be the same ordifferent, each represent: a hydrogen atom, a halogen atom, a hydroxygroup, a nitro group, a linear or branched (C₁-C₆) polyhaloalkyl group,a linear or branched (C₁-C₆) alkyl group, —OR₁₀, an amino group, asubstituted amino group optionally substituted by one or more arylgroups, heteroaryl groups, or linear or branched (C₁-C₆) alkyl groups or(C₃-C₆) cycloalkyl groups optionally substituted by a carboxylic acidgroup, by a carbonyl group, or by a group of formula —CONR₈R₉ or —NR₈R₉,—OPO(OH)₂, an acyl group, a carboxylic acid group, a sulfonyl group, asulfonamide group, or a cyano group; and R₆ is a hydrogen atom, a linearor branched (C₁-C₆) alkyl group, a (C₃-C₆) cycloalkyl group, or an arylor heteroaryl group optionally substituted in one or more positions withone or more of R₂, R₃, R₄, and R₅, each of which may be the same ordifferent, wherein the cancer is selected from the group consisting ofbreast cancer, lung cancer, and leukemia.
 2. A method of treating acancer in a patient, comprising administering to a patient in needthereof a therapeutically effective amount of a compound having theformula I:

or a pharmaceutically acceptable salt, ester stereoisomer, or solvatethereof, wherein: n is 1; W is O; X is O, NH or S; R₁ is H; R₂, R₃, R₄and R₅ are each H; and R₆ is phenyl or benzo[d][1,3]dioxolyl, each ofwhich may be optionally substituted with one or more substituentsindependently selected from the group consisting of hydroxy, methoxy,methyl, N(CH₃)₂, NH₂, —NHCOCH₃, fluorine, bromine, and chlorine, whereinthe cancer is selected from the group consisting of breast cancer, lungcancer, and leukemia.
 3. A method of treating a cancer in a patient,comprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound selected from the group consisting of:

and a pharmaceutically acceptable salt, ester, stereoisomer, or solvatethereof, wherein the cancer is selected from the group consisting ofbreast cancer, lung cancer, and leukemia.